Method and devices for preventing restenosis in cardiovascular stents

ABSTRACT

Described herein are devices and methods fabricating devices having nanostructures that allow adhesion or growth of one cell type, such as endothelial cells, more than another cell type, such as smooth muscle cells. In particular, stent covers having such nanostructures are described, and methods for fabricating these stent covers. Also described herein are methods for optimizing the nanostructures forming the devices.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No.60/952,818 filed on Jul. 30, 2007, entitled, “METHOD AND DEVICES FORPREVENTING RESTENOSIS IN CARDIOVASCULAR STENTS.”

INCORPORATION BY REFERENCE

All publications and patent applications mentioned in this specificationare herein incorporated by reference in their entirety, as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

BACKGROUND OF THE INVENTION

Stents have become the treatment of choice for a variety of blood vesseldiseases in humans and are in common use worldwide. From the beginningof their use, it is known that an unacceptable percentage of cardiacstents become blocked after installation. This restenosis is a serioushealth problem: a patient may be more seriously impaired after treatmentthan before. Restenosis occurs when smooth muscle cells in the bloodaggregate into clumps and cause the stent to become occluded.Drug-eluting coatings have been used to prevent clumping. Based oncurrent data, it now appears that these coatings are not a satisfactorysolution. For example, coated stents have been shown to cause bloodclots several years after installation (Brian Vastag, “Stents Stumble,”Science News, Jun. 23, 2007 Vol. 171, pp 394-395). A patient receiving acoated stent must use blood thinners to prevent formation of blood clotsthat may dislodge from the region of the stent and cause stroke or heartattack. If the blood thinning treatment is interrupted, the probabilityof stroke is greatly increased. Thus, coated stents have thereforeproved not to be a viable solution to the problem of stent restenosis.

It has recently been proposed that the restenosis of a stent is largelydetermined by whether the first layer of cells to grow on the surface ofa stent are endothelial cells or smooth muscle cells (Julio Palmaz,Lecture at SMST conference 2003, Asilomar Calif. Comments not includedin Proceedings of the conference). Described herein are stent coverswhich selectively enable endothelial cells to grow on their surface incomparison to smooth muscle cells, as well as methods of manufacturingand using them.

Other methods of preventing restenosis have been developed. For example,Palmaz describes a method using chemical affinities (resulting fromcharges on the surface of the stent) to regulate the growth ofendothelial cells versus smooth muscle cells. U.S. Pat. No. 6,820,676 toPalmaz et al. teaches the use of surface relief patterns correspondingto crystal boundaries in metal to the enhance growth of cells. However,these patterns are irregular in size and shape and so are not ideallyselective for a specific protein. In WO07078304A2, Dubrow et al. suggestusing fibers (e.g., nanofibers or nanowires) to form a substrate for usein various medical devices.

Stents using shape memory alloys (e.g., Nitinol) to form thin-film stentcovers are currently being developed. For example, US 2008/0161936 toFeller et al. describes a stent including a thin-film of shape memoryalloy having different porosities in the deployed and undeployedconfigurations. A stent cover is typically placed over a stent toprovide protection against debris that may be dislodged duringinstallation of the stent. Described herein are stents or stent coversincluding a thin film coating or surface that preferably selectsendothelial cells (e.g., from the blood stream) to grow on the insidesurface of a thin-film stent or stent cover compared to other cell types(e.g., smooth muscle cells). This may be accomplished by formingnanostructures on the inside of the stent or stent cover. Thesenanostructures may be formed of one or more layers, and may correspondto recurring patterns on endothelial cell membrane proteins. Endothelialcells that come in contact with the surface nanostructures mayselectively adhere to them. For example, endothelial cells (as opposedto smooth muscle cells) may ‘recognize’ the surface structure by patternmatching (or pattern recognition) and adhere. This pattern recognitionstep is a key element in many molecular biology processes. The devicesand methods described herein take advantage of this native molecularbiological process (e.g., cell-surface interactions) to influence theadherence of one type of cell (e.g. endothelial cells) in preference toother types (e.g. smooth muscle cells). Thus, surface nanostructures maybe used to selectively enhance adhesion of endothelial cells over smoothmuscle cells.

SUMMARY OF THE INVENTION

General methods of forming thin-film, nano-scale layered substratesappropriate for controlled cell growth are described herein. Inaddition, we describe particular applications of such nano-layeredcontrolled growth substrates. In particular, described herein areimplantable medical devices having nanostructures that allow adhesionand/or growth of one cell type, such as endothelial cells, more thananother cell type, such as smooth muscle cells. For example, stents andstent covers having such nanostructures are described. Methods forfabricating these devices are also described.

For example, described herein are methods of controlling the growth ofcells, including differential or preferential growth of cells, includingthe steps of patterning a substrate to have an organized nanostructurefor cell growth, and placing cells on the patterned substrate. Cells maybe regulated or controlled in any number of ways by the nanostructuredsurfaces described herein, including, as mentioned, differential growth(e.g., of various different cell types), cell guidance (e.g., axonguidance, cell migration, etc.), apoptosis, inhibition of cell growth,and the like.

A substrate may be patterned by: (a) layering a first nanostructuralmaterial at least partially over a substrate; (b) layering a secondnanostructural material at least partially over the layer from step a;(c) repeating steps a and b to form a plurality of alternating layers ofthe first nanostructural material and the second nanostructuralmaterial; (d) forming a patterned layer on the outer surface of theplurality of alternating layers; and (e) etching the nonstructuralmaterial to form a nanostructured surface. In some variations, the stepof layering the second nanostructural material comprises depositing ashape memory alloy at least partially over the first nanostructuralmaterial layer. The shape memory alloy may also be crystallized.

In general, these substrates are different from other cell culturesubstrates because they are formed using lithographic procedures to havesides or walls that are alternating layers or striated layers, ofnanostructural materials (e.g., metals, alloys, polymers, etc.) that canbe repeated patterns may layers thick. For example, one well or isletmay have walls comprising five, ten, fifteen, twenty, twenty-five, fiftyor even a hundred or more alternating layers of nanostructuralmaterials. Each layer (or sub-layer) may be extremely thin (e.g., on theorder of a few nm of thickness), and differ layers may have differentthicknesses. The geometry may be important. For example, not only thethickness of each layer (or sub-layer), but the overall shape of thewell or islet formed, as described in further detail below, may affectthe growth or development of the cells contacting them.

For example, a method of forming a stent cover having a nanostructureincludes the steps of forming a protective patterned layer on an outersurface of a sacrificial mandrel, layering a first nanostructuralmaterial at least partially over the protective patterned layer,depositing a shape memory alloy at least partially over thenanostructural material layer, crystallizing the shape memory alloy,removing the sacrificial mandrel, and etching the nonstructural materialto form a nanostructured surface surrounded by a shape memory alloy thinfilm.

Any appropriate shape memory alloy may be used, including anickel-titanium alloy (e.g., Nitinol), and the method may also includethe step of fenestrating the shape memory alloy.

The sacrificial mandrel may be formed of an etchable metal, such ascopper, that may be removed prior to etching the nanostructured layer. Aprotective patterned layer may be formed on the mandrel in order topattern the nanostructural layer by acting as a resist layer to protectit (or a portion of it) during processing to form the nanostructurallayer, particularly when the processing includes lithographic processingto form the nanostructural layer. The protective patterned layer mayitself be formed on the mandrel photolithographically. For example, theprotective patterned layer may be formed by coating a chromium layer onthe sacrificial mandrel, applying a photoresistive layer, and thenexposing it to a negative or positive of the desired protective pattern.For example, the protective patterned layer may be a checkerboardpattern to form islets of nanostructural material. In some variations, apattern of wells may be formed.

Any appropriate material may be used as the nanostructural material. Forexample, the nanostructural material may include metals (includingalloys), ceramics, polymers (including biopolymers), peptides (includingpolypeptides), nucleotides, composite materials, or the like. Thenanostructural material may be a core matrix that is doped withadditional materials. In some variations, the nanostructural materialmay be selected from the group consisting of: Ti, TiNi, Au, Ni, Al, Fe,Pt, Hf. Other materials having specific properties may be used,including those taught by Haushalter (U.S. Pat. No. 7,229,500). In somevariations, the nanostructured layer includes a plurality of layers(including sub-layers). For example, a second nanostructural materialmay be at least partially applied over the protective patterned layer.The first nanostructural material may be applied to a thickness ofbetween about 10 nm and about 500 nm. Similarly the secondnanostructural material may be applied to a thickness of between about10 nm and about 500 nm.

Also described herein are stent covers including nanostructures to whichendothelial cells adhere preferentially compared to smooth muscle cells.A stent cover may include a thin-film of a shape memory material, thethin film at least partially surrounding a nanostructural layer, whereinthe nanostructural layer comprises a layered nanoscale pattern having ashape and thickness that is selected so that endothelial cellspreferentially adhere to the nanostructural layer as compared to smoothmuscle cells.

As mentioned above, the shape memory material may be a nickel-titaniumalloy (e.g., Nitinol). The shape memory material may further comprisefenestrations.

In some variations, the nanostructural layer comprises one or more pairsof sub-layers wherein each sub-layer is between about 10 nm and about500 nm thick. The nanostructural layer may be at least partially made ofa metal selected from the group consisting of: Ti, TiNi, Au, Ni, Al, Fe,Pt, Hf, and may have a thickness of between about 10 nm and about 500nm. In some variations, the nanostructural layer comprises one or moresub-layers having a thickness of between about 10 nm and about 500 nm.Other materials having specific properties may be used, including thosetaught by Haushalter (U.S. Pat. No. 7,229,500).

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe claims that follow. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings of which:

FIG. 1A shows a layout of a patterned wafer and FIGS. 1B and 1Cillustrates examples of types of patterns for cell growth.

FIGS. 2A-2D show various etched patterns of Ti—Ni—Ti multi-layers on asubstrate.

FIG. 3 is a schematic diagram illustrating one variation of a method forforming a stent cover, as described herein.

FIG. 4A is a test wafer on which a nanostructure has been formed.

FIG. 4B is a detailed view of a region of the wafer of FIG. 2A.

FIGS. 4C and 4D are cross-sectional views showing nanostructures.

DETAILED DESCRIPTION OF THE INVENTION

Substrates, devices incorporating substrates, methods of formingsubstrates, and methods of forming devices including the substrates aredescribed herein. In general, these substrates may be useful for tissueengineering. Surfaces such as those described herein that includestructures having well-controlled shapes and sizes may provide means forcontrolling the behavior of individual cells and collections of cells(including tissues). Cells, and even tissues, may be controlled bycontrolling the arrangement of proteins.

Substrates including the patterned surfaces described herein (includingthose patterned using shape-memory materials) may provide surfaces onwhich controlled cell growth is enabled. These surfaces may beconfigured to select for cell type (e.g., enhancing the growth ofcertain cells compared to other cells). The structure of the surface maybe the primary factor in regulating the cell behavior, rather than thechemical or material properties of the surface, as is commonly believed.The structure of the surface may also help manipulate the proteins onthe surface of the cells and/or in the intracellular compartments of thecell.

The surface structures described herein are controlled over thedimension of the nanometer-to-micron scale. This scale (including thescale of the thickness of the layers) may be selected and controlled ina range of tens of nanometers to hundreds of nanometers, which iscomparable to the size of most proteins. The scale of the micromachinedstructures (e.g., microns to tens of microns) is commensurate with thesize of most cells. Thus, the methods described herein may allow theselection of proteins, which may be specific to certain types of cells,and of cell size and shape, which may also be specific to certain celltypes. Taken together, these two selection methods may be much strongerthan selection by either method alone. Unlike existing methods, whichrely primarily on abrasion and have little or no control of theshape/size/pattern of the structures formed, these methods may createordered and predictable patterns of surface structures having apredetermined shape and size. Typically, the layers forming the surfacesdescribed herein are controlled to one percent or better.

The methods described herein may be used to create surfaces havingorganized arrangements of shapes. The shapes may be chosen based on theeffect that the shape will have on cell behavior. For example, anelongated shape may encourage cell division (i.e., mitosis). A roundshape may cause cells to undergo apoptosis (i.e., cell death). Othershapes may have different effects, such as encouraging migration orsecretion. The thickness or shapes on the substrates described hereinmay also modify the expression of proteins on the surface of cells. Forexample, ledges that are of the same thickness dimension as a particularprotein may enable formation of protein structures that do not occur innature. A protein may bind to the surface in such a way that it iselongated rather than folded. If many proteins lie adjacent to eachother in a stretched-out configuration, they may resemble (and may form)a novel crystal structure.

As mentioned, these substrates may be useful for growing tissues andorgans. The location and spatial arrangement of individual cells in amulti-cell pattern may provide the scaffolding for development ofspecialized tissues.

FIGS. 1A-2D illustrate exemplary substrates, including patterns. Thesenano layered substrates may provide nanometer scale structures for usein cell growth experiments, for clinical cell growth, and for virtuallyany cell culture purpose. Furthermore, such surfaces may be included aspart of a medical device (e.g., implant) or other medical interface, asdescribed in greater detail below.

For example, FIG. 1A shows an exemplary pattern that may be formed on asilicon wafer or slide. In this example a thin film consisting ofalternating layers of two materials (e.g., TiNi and Ti) are formed on asilicon die (slide). In this example, each layer may be vacuum sputterdeposited to a thickness of between 10 and 250 nm. The thickness may betightly controlled (e.g., a thickness of 10 nm for that layer may beapproximately uniform at 10 nm), or it may be allowed to increase (e.g.,ramp increase) across the layer. The actual thickness applied to thelayer may be selected from the range (e.g., 10 to 250 nm) so that thefinal thickness is precisely known. Further, the number of layers formedof each material (e.g., TiNi, Ti, etc.) may be controlled. Typicallymultiple layers (e.g., 5 layers of each material, 10 layers of eachmaterial, 20 layers of each material, 30 layers of each material, etc.)may be chosen. The number of layers and the thickness of the layers mayhelp form the complete thickness of the wells. As described in greaterdetail below, the side surfaces formed by these alternating layers(which may provide a striated or stratified side surface) may furthercreate an ordered surface for cell or tissue growth. The differentlayers may be alternated in any desired pattern.

After deposition, wells may be created in the thin film byphotolithographic techniques, including chemical etching. Wells may bebetween 1 and 10 microns in extent. The wells may have varying shapesand sizes to provide a variety of attachment conditions for celldevelopment. FIG. 1A illustrates one example of a layout of a pattern ona wafer as viewed from the “macro” (naked eye) scale. Each horizontalline in this example indicates a row of one type of pattern. FIGS. 1Band 1C show different patterns that may be used. The images shown inFIGS. 1B and 1C are enlarged to show the shapes.

For example, the wafer shown in FIG. 1A may be divided up into 20 mm×20mm slides. Each slide has approximately 50 rows of approximately 20wells. In FIG. 1B, the rows (which may be included in the slide) haverectangular openings, and in FIG. 1C the rows five circular openings.The scale of these openings is between about 10 and about 400 microns indiameter. For example, the circles in FIG. 1C range in diameter from 10to 200 microns (e.g., 10, 20, 40, 100, and 200 microns).

FIG. 2A-2D shows images of patterns etched in TiNi—Ti multi-layers on asilicon wafer, as briefly described above. For example, FIG. 2A shows arow of repeated rectangular openings. In one example, four groups ofrectangular openings may be patterned. For example, the groups may be 10microns wide by 100, 200, 300, and 400 microns long, 20 microns wide by100, 200, 300, and 400 microns long, 40 microns wide by 100, 200, 300,and 400 microns long, and 80 microns wide by 100, 200, 300, and 400microns long. The nanolayered substrates shown in FIGS. 2A-2C illustratepatterning of twenty alternating layers of Ti and TiNi deposited on asilicon or silicone oxide wafer surface to form a thin film that is 2microns thick. The patterns of circles or rectangles can then be etchedthrough the film. FIG. 2D shows an SEM image of a portion of an etchedshape. The “walls” forming the shape are formed of the multiple layersof material, in this example, alternating layers of TiNi and Ti. Thewalls may be more or less ‘steep’ by controlling the etching or removalof the materials, and/or masking or protecting etched layers.

This general technique for forming a surface by patterning a substratemay be applied to substrates having non-flat (e.g. curved, bent,cylindrical, etc.) surfaces. For example, a medical implant, such as acatheter, stent, or the like, may include a nanolayered surface that ispatterned appropriately to encourage and/or discourage cell growth orother cell behaviors.

Described below are stent covers and methods of forming stent covershaving a preferentially patterned surface. For example, a method fordetermining a pattern that preferentially favors endothelial celladhesion over smooth muscle cell adhesion is described. Although thesemethods are described with particular reference to stent covers, theymay also be applied to the surface (particularly the “inner” or luminalsurface) of a stent itself, or other structures, including other medicaldevices. Further, these methods and devices are not limited to surfacesfavoring endothelial cell growth over smooth muscle cells. Surfaceshelping to control (including differential growth, inhibition, cellguidance, apotosis, etc.) of any appropriate cell type may be formedusing the methods and systems described herein. The patterned surfacesdescribed herein may also be referred to as nanostructured surfaces,nanostructured layers, or nanotextured surfaces.

One variations of the nanostructured surfaces descried herein includespatterned stent covers.

Method of Forming a Patterned Stent Cover

Referring to FIG. 3, a stent cover may be formed having a patternpreferential for adhesion of endothelial cells compared to smooth musclecells.

The patterned stent covers described herein may be patterned in theappropriate size scale for preferential adhesion of endothelial cells.It is desired that the size be highly selective, providing a majoradvantage for endothelial cells to grow relative to smooth muscle cells.For example, the scale of the patterns described herein may be in thenanometer range (e.g., between 0.1 and 500 nm). It is believed that aregular pattern of 200 nm balls creates a surface that is selective forendothelial cells. (Derick C. Miller, Journal of Biomedical MaterialsResearch, Part A, 81A, Issue 3, 678-684: cited in “Nanotechnology forHealing Damaged Vascular Tissues,” Thomas Webster,www.scitizen.com/screens/blogPage/viewBlog/, last viewed Jul. 20, 2007).

A nanostructured surface may be formed as a layer created on the insideof the tubular stent cover by forming a protective pattern (e.g., bymasking or otherwise protecting the material), then depositing a layeror layers of material that will form the nanostructural surface, andforming the outer shape memory material around the protective patternand deposited layers. Once the shape memory material has beensufficiently prepared (e.g., crystallized), the mandrel may be removed,and the nanostructural surface may be formed by etching the patternusing microlithography to etch around the protected material of theprotective pattern.

FIG. 3 describes one exemplary method for forming a stent cover having ananostructural inner surface that is selective for endothelial cellsover smooth muscle cells. In FIG. 3, a sacrificial mandrel is firstprepared 101. In general, a sacrificial mandrel is a form that may beremoved (e.g., by etching, erosion, etc.) once the layers of the stentcover have been formed around it. Thus, the sacrificial mandrel may besimilar is shape and dimension to the outer surface of a stent withwhich the cover will be used. The material of the sacrificial mandrel istypically removable or etchable. For example, the sacrificial mandrelmay be formed of copper. See, for example, U.S. Pat. No. 6,923,829 Boyleet al. These procedures are well known in MEMS technology.

A protective pattern may then be formed around the outside of themandrel 103. As mentioned above, the protective pattern (or protectivelayer) at least partially forms the pattern of the nanostructure byselectively protecting the material forming the nanostructural surfacefrom etching. For example, the protective pattern may be made from athin layer of chromium, e.g., 10-100 nm thick, that is laid down on acylindrical substrate 103 and formed into a desired pattern. Theprotective pattern may be formed by photolithographic techniques, asknown in the art. For example, the chromium layer may be coated with aphotoresist material and patterned by exposing it to negative (orpositive) of at least a portion of the nanostructural layer followed byremoval of the unexposed photoresist, etching to remove unprotectedchromium, and removal of the photoresist layer. In one variation thechromium layer forms the shape and pattern of islets of thenanostructural surface. For example, the chrome layer may be patternedwith a checkerboard design, so that the islets forming the nanostructurewill be columnar islets having a square cross-section (see, e.g., FIGS.4A-4C). After forming the protective pattern, the material forming thenanostructural surface layer may be applied over the protective pattern105.

For example, alternating layers of material may be deposited over theprotective pattern, as taught by Haushalter (U.S. Pat. No. 7,229,500).These layers may be referred to as sub-layers of the nanostructuredsurface, and will form the nanostructured surface. Any appropriatematerial may be layered on the protective pattern to form thenanostructured surface. In particular, materials may be chosen based ontheir potential for encouraging adhesion and/or growth of endothelialcells selectively, compared to smooth muscle cells. For example, thematerial may be a metal or alloy, including (but not limited to): Ti,TiNi, Au, Ni, Al, Fe, Pt, and Hf. Other materials having specificproperties may be used, including those taught by Haushalter (U.S. Pat.No. 7,229,500). In some variations, the material is a doped materialthat includes a predetermined amount of some additional component. Forexample, the material forming a sub-layer of the nanostructured surfacemay be a metal or alloy that is doped with one or more transitionelements, alkali (rare earth) metals, colloids, etc.

In some variations the layer of material forming the nanostructuredsurface includes a plurality of sub-layers of different materials. Forexample, the nanostructured surface may include repeated layerscomprising two or more sub-layers. The material of each sub-layer may bedifferent. The material properties of each sub-layer or layer may bedifferent (e.g., hydrophobic/hydrophilic; +/−charge, etc.), inparticular, there may be a difference in their rate of removal by aparticular etchant. Thus, the materials forming the nanostructuredsurface may form one or more interfaces that can enhance attachment toendothelial cells, or inhibit attachment of smooth muscle cells. In somevariations, the nanostructured surfaces include alternating sub-layersthat may be differently etched to include recessed portions.

As mentioned, the sub-layers forming a nanosurface may be repeated. Forexample, a nanosurface may include a repeated motif of two or moresub-layers (e.g., sub-layer A and sub-layer B), that are repeated ntimes, so that the nanosurface comprises n layers of AB (or BA,depending on the order). The thickness of sub-layer A and sub-layer Bmay be uniform in each repeated layer, or the thicknesses may vary.

The dimensions of the nanostructured surface may be set by thecombination of the pattern (e.g., the protective pattern), the thicknessof the material(s) applied to form the nanostructured surface, and theetching used to form the nanostructural layer. In general, thenanostructured surface has dimensions that are within the nanometerscale (e.g., between 0.1 nm and 500 nm). For example, the nanostructuredsurface may be a repeated pattern of nanostructural “islets” having apredetermined depth (e.g., between 10 and 500 nm) and cross-sectionalprofile which matches the shapes formed of the protective pattern. Forexample, the islets may have square, rectangular, triangular, circular,oval, etc. shape. The predetermined depth of the islets may bedetermined by the thickness of the material(s) applied to the protectivepattern on the mandrel (e.g., the thickness may be between 10 and 500nm). The spacing between the islets may also be determined by theprotective pattern (e.g., individual islets may be separated by between10 and 500 nm, such as about 100 nm). In some variations, thenanostructure surface includes one or more wells etched to a shapeand/or dimension as desired. For example, the wells may have a round,oval, rectangular, or other shape, and may be between about 10 and 500nm deep.

As mentioned, the shape, dimension and spacing of the islets/wellsforming the nanostructure may be specifically chosen so that adhesion orgrowth of one cell type (e.g., endothelial cells) is favored incomparison to another cell type (e.g., smooth muscle cells). Thus, theshape, depth and arrangement of the islets/wells may be determinedexperimentally, as described in the examples below.

Returning now to FIG. 3, after the appropriate thickness of materialforming the nanostructured surface has been applied 105, a layer ofshape memory material is then applied 107. Any appropriate shape memorymaterial may be used, particularly a shape memory alloy such as a nickeltitanium alloy. One preferred material is Nitinol. The shape memorymaterial is deposited as a thin film. When the desired thickness of thinfilm has been deposited, the material may then be heat-treated tocrystallize 109. In some variations, the thin film layer shape memorymaterial is then fenestrated (e.g., using standard MEMSphotolithography). U.S. Pat. No. 6,533,905 to Johnson et al. describesone method of applying (e.g., by vacuum deposition including sputtering)a TiNi shape memory alloy as mentioned above, and a patterned stentcover may be made using some of the teachings described therein. Forexample, U.S. Pat. No. 6,533,905 may be exploited to make a tubularstent cover.

The thin layer of shape memory material may also be patterned. Forexample, the shape memory material may be fenestrated 111. Fenestrationsmay be biologically desirable (e.g., allowing access or in-growth).

The sacrificial substrate (e.g., the mandrel) may be removed, leaving acylinder of thin film material (e.g., shape memory material) with aprotective layer (e.g., chromium) and the layer(s) of materials thatwill form the nanostructured surface inside 113 the cylinder. Thematerial forming the nanostructured layer(s) can be patterned byexposure to appropriate etchants 115.

The finished stent cover formed by the method of FIG. 3 is afenestrated, tubular, thin film of shape memory material (e.g.,Nitinol), inside of which is a surface consisting of nanostructures thathave appropriately structures (e.g., layered islets) to selectively growa layer of cell, including endothelial cells, preferentially to anothertype of cell, such as smooth muscle cells in the blood stream.

In general, materials (particularly the material or materials formingthe nanostructured surface) must be selected for compatibility of theother steps of the method. For example, these materials must surviveheat treatment without damage. Materials used must be compatible withdeposition and processing of TiNi film, including crystallization at500° C. Further, the etchants must be highly selective.

An assumption is made that there are different proteins in the cellmembranes of smooth muscle cells than on endothelial cells. Theexperimental fact that endothelial cells grow preferentially on layersformed from spheres 200 nanometers in size appears to support thisassumption (Thomas Webster, Nanotechnology for healing damaged vasculartissue, Nanomedicine Laboratories, Brown University, Providence R.I.,www.scitizen.com).

A further assumption is that a repeating pattern of layers having anappropriate distance of repetition, each layer consisting of two or moresub-layers differing in some quality, will also act to enhance growth ofendothelial cells. Since the appropriate distance is not known, it maybe determined by experiment, as mentioned above. The examples belowoutline one method in which these parameters may be determinedexperimentally.

EXAMPLES Optimizing a Nanoscale Pattern

As used here, the term “nanostructural pattern” may refer to a surfaceor layer having nanostructural features. In general, this is a surfacehaving one or more repeated motifs of nanoscale structures. Thesenanoscale structures may be repeated “islets” etched from the one ormore layers of material (or sub-layers) forming the nanostructurallayer. Thus, the nanostructural layer may be a plurality of columnarislets separated from each other by a “street” or channel having apredetermined depth. A cross-section through an islet may be square,rectangular, circular, etc. The sides of an islet may present stratifiedlayers made up of different materials, and the thickness (depth) of eachlayer (as well as the material from which the layer is made) may bechosen to select one type of cell over another type of cell (e.g.,endothelial cells over smooth muscle cells). These islets may thereforepresent a larger surface area of interface boundaries between differentmaterials which cells may contact. Certain cell types may preferentiallyadhere to some interface regions.

One or more nanostructural patterns that are selective for endothelialcells over smooth muscle cells may be determined experimentally usingthe methods and devices described herein. For example, U.S. Pat. No.7,229,500 to Haushalter et al. describes a method of enhancing thecrystallization of certain species of proteins by providingheterogeneous nucleation sites consisting of multiple nanometer scalethickness layers with alternating characteristics. The methods of theU.S. Pat. No. 7,229,500 may be adapted to form nanostructural patternsthat may be tested to determine nanostructural layers (sub-layers) orpatterns that preferentially support endothelial cells over smoothmuscle cells. Nanometer scale, regular patterns of alternating layersand/or materials can be generated and systematically examined forinteraction with both endothelial and smooth muscle cells, to determinewhich one or more regions of specific patterns may act as scaffolds forspecific types of cell (e.g. endothelial cells) by causing their cellmembrane proteins to adhere to the surface more strongly than othertypes of cells (e.g. smooth muscle cells). Successful application ofthis invention may result in a continuous layer of endothelial cellsbeing formed on the surface of the stent cover.

In some variations, a library of different nanostructures (islets and/orwells) may be formed of layers of selected materials and thicknesses, sothat the optimum thickness and materials can be determined to beselective for endothelial cells. FIGS. 4A-4C illustrates one variationof this. Initial experiments may be performed to determine whetherendothelial cells can be separated from smooth muscle cells by gradedthickness of metal on substrate. In one variation of this experiment,selectivity may be determined by varying the size scale.

Endothelial and smooth muscle cells may be grown on substrates withraised islets of graded varying thickness or with wells or depressionsof various depths. The substrates may be inspected to see if cells growpreferentially on islets of a particular thickness and/or wells or aparticular depth. The experiment may be performed by first preparing thesubstrate. In this example, the substrate is a wafer 201 onto whichislets have been formed. The islets or wells (nanostructures) may beformed by sputter depositing one or more metal layers. The thickness ofthe metal layer may vary continuously from a few nanometers on one edgeof wafer to about 500 nanometers on the other edge. After coating withlayers of metal(s), the islets/wells may then be formed by patterningthem by photolithography, and chemically milling them to produce regularpatches or islets of metal, as shown in FIG. 4B, which is a close-upview of a region of the wafer 201 and 2C, which shows a cross-sectionthrough a wafer. In this example, islets 203 near one edge of the waferare about 500 nm, and islets near other edge of the wafer are about 10nm. Patterns in a particular region of a wafer may be kept uniform inlength and width so that this is not a variable. For example, thecross-sectional shape of the islets on a particular wafer may beconstant, even though the depth is varying. The size of islets may bevaried from one wafer to another to test the effect of cross-sectionalsize. The cross-sectional shape (e.g., circular, polygonal—e.g.,triangular, rectangular, square, etc.) may also be varied between wafers(or on a single wafer) to determine an effect. FIG. 4C illustrates across-section through a wafer (not to scale) showing exemplary isletshaving different thicknesses, e.g., from 10 nm (209) to 500 nm (207).

Different islet materials may also be tested. For example, differentmetals may be tested, including (but not limited to): Ti, TiNi, Au, Ni,Al, Fe, Pt, and Hf. In some variations, other materials may be used. Insome variations, the layers of material may be doped with one or moremolecules. For example, there are known cell-adhesion proteins that maybe incorporated as part of the layered material. Other materials includetransition elements, alkali or alkaline/rare earth materials, oxyacids,nitrates, acetates, sulfates, colloids, etc.

To test, pairs of wafers may be exposed to two different cell types, ora single wafer may be exposed to two different cell types (e.g., thewafer may be divided in half). For example, one wafer (or half a wafer)may be exposed to endothelial cells, while the other wafer (or half ofthe wafer) is exposed to smooth muscle cells. After an appropriate time,the wafers may be inspected to determine if there is a significantdifference in the way the two types of cells adhere and/or grow. Thismethod may help answer the question as to whether cells growpreferentially on a specific thickness of material, as well as whichmaterials may be preferred. In particular, this type of experiment mayhelp determine if there is a thickness and/or material to whichendothelial cells adhere or grow better than smooth muscle cells.

The test wafers described herein may be formed on a glass substrate. Forexample, a 4-inch wafer having an oxidized surface (to present a smoothglass surface) may be used. A pair of wafers may each be identicallydeposited with the metal (or other material) in a thickness gradientfrom one side of the wafer to the other. For example, the thickness maybe varied from 500 to 10 nanometers. Afterwards, the islets and/or wellsmay be patterned. In one variation, three patterns of islets/wells(having different cross-sectional sizes) may be formed per wafer. Forexample, a square profile islet having a length and width of 1 mm, 0.5mm, or 0.1 mm may be formed on each wafer. The spacing between eachislet (the street) may be about 100 nm wide. The islets may be patternedby photolithography using a prepared mask exposed to the coated surfaceof the wafer after applying a layer of photoresist. The exposedphotoresist may then be removed, and the wafer etched. Afterwards, thewafer (including the formed islets) may be cleaned to remove allchemicals (etchant, etc.) from the wafer.

Cell growth or adhesion on the wafers may be tested in any appropriateway. For example, cell growth may be examined after incubating withcells or after exposing to flows of cells. Wafers can then be examinedfor successful growth, especially growth at a particular metalthickness. Statistics (cell counts, etc.) may be taken to quantify anyeffects.

The sensitivity of these experiments may be enhanced as part of a secondphase of experiments. Selectivity can be increased by makingnanostructures (e.g., islets, wells, etc.) having multiple layers, asshown in FIG. 4D. Different materials may be layered onto the wafer inalternating layers 211, 213, 215, 217. Materials having contrastingproperties (e.g., hydrophilic/hydrophobic, charged/uncharged,+/−charges, doped/undoped, and/or different etching rates) may be ofparticular interest. Multiple-layered islets/wells may be formed similarto the method just described above, and similarly tested. In addition totests against isolated cells, materials may be tested in an animalmodel. For example, rather than using a glass wafer as the testsubstrate, the substrate may be a nickel-titanium material. Thus, atesting stent or stent cover may be formed having varyingcharacteristics for the nanostructures, as briefly described above, andimplanted into an animal model for testing. This test stent cover orstent may be retrieved after an appropriate time, and histologicallyexamined to determine which regions (corresponding to whichnanostructures) had desirable properties with respect to endothelialversus smooth muscle cells.

The nanostructured layer may be formed of multiple layers or sub-layersof material, as described above and illustrated in FIG. 4D. In somevariations, the nanostructured surface may be formed of multiplerepeated sub-layers. For example, two layers can be repeated to form alayer that may be etched to form the nanostructured surface. Thethicknesses of the two sub-layers may be varied in two gradients acrossthe surface of the substrate. For example, the thickness of the firstsub-layer can decrease from the left to the right of the substrate. Thethickness of the second sub-layer can decrease from the back to thefront of the substrate. The overall thickness of the nanostructuredsurface (the depth of each islet) may be kept approximately constantacross the substrate (e.g., by increasing the number of layers applied).This substrate may be used to test the effect of the thicknesses of eachsub-layer on cell adhesion and growth, as described above.

In addition to the thickness of the nanostructure-forming layer(s), theshape of the nanostructure(s) may also be varied. Differential etchingmay be used to form a superlattice having different interlayer spacing.For example, one or the sub-layers forming the nanostructured surfacemay be etched more than the other sub-layer(s). This results in ananotextured 3-D surfaces in which one of the sub-layers is undercutcompared to the other sub-layer, forming indentations, pockets, ledgesand/or terraces along the surface. The amount of relative etching ofeach sub-layer can also be varied to determine the effect on celladhesion and growth, as described above. For example, by varying theetching times and/or etch rates, different amounts of surface etchingmay be achieved.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it is readily apparent to those of ordinary skill in theart in light of the teachings of this invention that certain changes andmodifications may be made thereto without departing from the spirit orscope of the appended claims.

1. A method of forming a stent cover having a nanostructure, the methodcomprising: forming a protective patterned layer on an outer surface ofa sacrificial mandrel; layering a first nanostructural material at leastpartially over the protective patterned layer; depositing a shape memoryalloy at least partially over the nanostructural material layer;crystallizing the shape memory alloy; removing the sacrificial mandrel;and etching the nonstructural material to form a nanostructured surfaceat least partially surrounded by shape memory alloy.
 2. The method ofclaim 1, wherein the shape memory alloy is a nickel-titanium alloy. 3.The method of claim 1, further comprising fenestrating the shape memoryalloy.
 4. The method of claim 1, wherein the sacrificial mandrel isformed of copper.
 5. The method of claim 1, wherein the protectivepatterned layer is formed by photolithographically patterning a chromiumlayer on the sacrificial mandrel.
 6. The method of claim 1, whereinfirst nanostructural material is selected from the group consisting of:Ti, TiNi, Au, Ni, Al, Fe, Pt, Hf.
 7. The method of claim 1, furthercomprising depositing a second nanostructural material at leastpartially over the protective patterned layer.
 8. The method of claim 1,wherein the first nanostructural material is applied to a thickness ofbetween about 10 nm and about 500 nm.
 9. The method of claim 7, whereinthe second nanostructural material is applied to a thickness of betweenabout 10 nm and about 500 nm.
 10. The method of claim 1 wherein thefirst nanostructural material comprises one or more pairs of sub-layerswherein each sub-layer is between about 10 nm and about 500 nm thick.11. A stent cover including nanostructures to which endothelial cellsadhere preferentially compared to smooth muscle cells, the stent covercomprising: a thin-film of a shape memory material, the thin film atleast partially surrounding a nanostructural surface, wherein thenanostructural surface comprises a layered nanoscale pattern having ashape and thickness that is selected so that endothelial cellspreferentially adhere to the nanostructural surface as compared tosmooth muscle cells.
 12. The device of claim 11, wherein the layerednanoscale pattern comprises a plurality of repeated sub-layers.
 13. Thedevice of claim 11, wherein the shape memory material is anickel-titanium alloy.
 14. The device of claim 11, wherein the shapememory material further comprises fenestrations.
 15. The device of claim11, wherein the nanostructural surface comprises one or more pairs ofsub-layers wherein each sub-layer is between about 10 nm and about 500nm thick.
 16. The device of claim 11, wherein the nanostructuralsurfaced is at least partially made of a metal selected from the groupconsisting of: Ti, TiNi, Au, Ni, Al, Fe, Pt, Hf.
 17. The device of claim11, wherein the nanostructural surface has a thickness of between about10 nm and about 500 nm.
 18. The device of claim 11, wherein thenanostructural surface comprises one or more sub-layers having athickness of between about 10 nm and about 500 nm.
 19. A method ofcontrolling the growth of cells, the method comprising: patterning asubstrate to have an organized nanostructure for cell growth by: a.layering a first nanostructural material at least partially over asubstrate; b. layering a second nanostructural material at leastpartially over the layer from step a; c. repeating steps a and b to forma plurality of alternating layers of the first nanostructural materialand the second nanostructural material; d. forming a patterned layer onthe outer surface of the plurality of alternating layers; and e. etchingthe nonstructural material to form a nanostructured surface; and placingcells on the patterned substrate.
 20. The method of claim 19, whereinthe step of layering the second nanostructural material comprisesdepositing a shape memory alloy at least partially over the firstnanostructural material layer.
 21. The method of claim 20, furthercomprising crystallizing the shape memory alloy.